Bicyclic sphingosine 1-phosphate analogs

ABSTRACT

Compounds that have agonist activity at one or more of the S1P receptors are provided. The compounds are sphingosine analogs that, after phosphorylation, can behave as agonists at S1P receptors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. 111(a) ofInternational Application No. PCT/US2007/003645, filed on Feb. 9, 2007,which claims priority to Provisional Application Ser. Nos. 60/771,789,filed Feb. 9, 2006, 60/855,960, filed Nov. 1, 2006 and 60/860,694, filedNov. 21, 2006, the disclosures of which are incorporated by reference intheir entirety.

US GOVERNMENT RIGHTS

This invention was made with United States Government support underGrant No. RO1 GM 067958 awarded by the National Institutes of Health.The United States Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that evokesa variety of cellular responses by stimulation of five members of theendothelial cell differentiation gene (EDG) receptor family. The EDGreceptors are G-protein coupled receptors (GPCRs) and on stimulationpropagate second messenger signals via activation of heterotrimericG-protein alpha (G_(α)) subunits and beta-gamma (G_(βγ)) dimers.Ultimately, this S1P-driven signaling results in cell survival,increased cell migration and, often, mitogenesis. The recent developmentof agonists targeting S1P receptors has provided insight regarding therole of this signaling system in physiologic homeostasis. For example,the immunomodulator, FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]propane1,3-diol), that following phosphorylation, is an agonist at 4 of 5 S1Preceptors, revealed that enhancing S1P tone influences lymphocytetrafficking. Further, S1P type 1 receptor (S1P₁) antagonists causeleakage of the lung capillary endothelium, which suggests that S1P maybe involved in maintaining the integrity of the endothelial barrier insome tissue beds.

Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that evokesa variety of cellular responses by stimulation of five members of theendothelial cell differentiation gene (EDG) receptor family.

Sphingosine-1-phosphate (S1P) has been demonstrated to induce manycellular processes, including those that result in platelet aggregation,cell proliferation, cell morphology, tumor-cell invasion, endothelialcell chemotaxis and angiogenesis. For these reasons, S1P receptors aregood targets for therapeutic applications such as wound healing andtumor growth inhibition.

Sphingosine-1-phosphate signals cells in part via a set of Gprotein-coupled receptors named S1P₁, S1P₂, S1P₃, S1P₄, and S1P₅(formerly EDG1, EDG5, EDG3, EDG6 and EDG8). The EDG receptors areG-protein coupled receptors (GPCRs) and on stimulation propagate secondmessenger signals via activation of heterotrimeric G-protein alpha(G_(α)) subunits and beta-gamma (G_(βγ)) dimers. These receptors share50-55% amino acid sequence identity and cluster with three otherreceptors (LPA₁, LPA₂, and LPA₃ (formerly EDG2, EDG4 and EDG7) for thestructurally related lysophosphatidic acid (LPA).

A conformational shift is induced in the G-Protein Coupled Receptor(GPCR) when the ligand binds to that receptor, causing GDP to bereplaced by GTP on the α-subunit of the associated G-proteins andsubsequent release of the G-proteins into the cytoplasm. The α-subunitthen dissociates from the βγ-subunit and each subunit can then associatewith effector proteins, which activate second messengers leading to acellular response. Eventually the GTP on the G-proteins is hydrolyzed toGDP and the subunits of the G-proteins reassociate with each other andthen with the receptor. Amplification plays a major role in the generalGPCR pathway. The binding of one ligand to one receptor leads to theactivation of many G-proteins, each capable of associating with manyeffector proteins leading to an amplified cellular response.

S1P receptors make good drug targets because individual receptors areboth tissue and response specific. Tissue specificity of the S1Preceptors is desirable because development of an agonist or antagonistselective for one receptor localizes the cellular response to tissuescontaining that receptor, limiting unwanted side effects. Responsespecificity of the S1P receptors is also of importance because it allowsfor the development of agonists or antagonists that initiate or suppresscertain cellular responses without affecting other responses. Forexample, the response specificity of the S1P receptors could allow foran S1P mimetic that initiates platelet aggregation without affectingcell morphology.

Sphingosine-1-phosphate is formed as a metabolite of sphingosine in itsreaction with sphingosine kinase and is stored in abundance in theaggregates of platelets where high levels of sphingosine kinase existand sphingosine lyase is lacking. S1P is released during plateletaggregation, accumulates in serum, and is also found in malignantascites. Reversible biodegradation of S1P most likely proceeds viahydrolysis by ectophosphohydrolases, specifically the sphingosine1-phosphate phosphohydrolases. Irreversible degradation of S1P iscatalyzed by S1P lyase yielding ethanolamine phosphate and hexadecenal.

Currently, there is a need for novel, potent, and selective agents thatare agonists of the S1P receptor having enhanced potency, selectivity,and oral bioavailability. In addition, there is a need in the art foridentification of, as well as the synthesis and use of such compounds.The present invention satisfies these needs.

SUMMARY

The present invention provides in one aspect compounds that have agonistactivity at one or more of the S1P receptors. The compounds aresphingosine analogs that, after phosphorylation, can behave as agonistsat S1P receptors. Accordingly, there is provided a compound of formulaI:

wherein X¹, Y¹ and Z¹ are independently O, CR^(a), CR^(a)R^(b), N,NR^(c), or S; R¹ is hydrogen, halo (C₁-C₁₀)alkyl, or (C₁-C₁₀)alkoxy; R²is hydrogen, halo, (C₁-C₂₀)alkyl, (C₁-C₂₀)alkoxy; (C₂-C₂₆)alkoxyalkyl;(C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl, (C₃-C₁₂)cycloalkyl, (C₆-C₁₀)aryl,(C₇-C₃₀)arylalkyl, (C₂-C₁₀)heterocyclic, (C₄-C₁₀)heteroaryl, or R² canbe a group having formula II, III, IV, V, or VI:

R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are independently O, S, C, CR¹⁵,CR¹⁶R¹⁷, C═O, N or NR¹⁸; R¹⁵, R¹⁶ and R¹⁷ are independently hydrogen,halo, (C₁-C₁₀)alkyl, (C₁-C₁₀)alkyl substituted with halo, hydroxy,(C₁-C₁₀)alkoxy, or cyano; and where R¹⁸ can be hydrogen or (C₁-C₁₀)alkyland at least one of R¹⁰, R¹¹, R¹², R¹³, or R¹⁴ is a heteroatom (O, S orN); Z² is (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, substituted alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₆-C₁₀)aryl, alkyl substituted aryl,(C₇-C₁₆)arylalkyl, or aryl substituted arylalkyl; wherein the alkylgroups of Z² are optionally substituted with 1, 2, 3, or 4 groupsindependently are halo, (C₁-C₁₀)alkoxy or cyano;

indicates one or more optional double bonds; Y² is O, C═O, or CH₂; W¹ isa bond or —CH₂—CH₂—CH₂—; W² is a bond or —CH₂— and m is 1, 2, or 3, or(C═O)(CH₂)₁₋₅ and m is 1. Each

represents an optional double bond; n is 0, 1, 2, or 3, and q is 0, 1,2, or 3.

R³ is hydrogen, (C₁-C₁₀)alkyl, or (C₁-C₁₀)alkoxy; and R⁴ is hydroxyl(—OH), phosphate (—OPO₃H₂), phosphonate (—CH₂PO₃H₂), oralpha-substituted phosphonate; R^(c) is hydrogen, or (C₁-C₁₀)alkyl.R^(a), R^(b), and R^(c) are independently hydrogen, or (C₁-C₁₀)alkyl.

The alkyl groups of R¹ can be optionally substituted with 1, 2, 3, or 4substituent groups, where the substituent groups independently are halo,(C₁-C₁₀)alkoxy or cyano. Any of the alkyl, alkenyl, alkynyl, cycloalkyl,aryl, heterocyclic, or heteroaryl groups of R² are optionallysubstituted with 1, 2, 3, or 4 substituent groups, where the substituentgroups independently are oxo (═O), imino (═NR^(d)), (C₁-C₁₀)alkyl,(C₁-C₁₀)alkoxy, or C₆-aryl, or wherein one or more of the carbon atomsin the R² alkyl groups can be independently replaced with non-peroxideoxygen, sulfur or NR^(c); the alkyl groups of R³ are optionallysubstituted with 1, or 2 hydroxy groups; and R^(d) is hydrogen, or(C₁-C₁₀)alkyl. The invention includes pharmaceutically acceptable saltsor esters of the compounds of formula I.

In another aspect, the invention provides phosphate esters havingformula VII.

In another aspect, the invention provides pro-drugs of the compounds offormula I. In another aspect, the invention also provides compounds offormula I or pharmaceutically acceptable salts or esters thereof for usein medical therapy.

In another aspect, the present invention provides a method forinhibiting angiogenesis in a tumor, comprising contacting the cancerouscells with an effective amount of a compound of formula I, or apharmaceutically acceptable salt thereof.

In another aspect, the invention provides a method for modulating theimmune system by altering lymphocyte trafficking for treatment ofautoimmune diseases or prolongation of allograft transplant survival,said method comprising administering an effective amount of at least onecompound of formula I to a subject in need thereof.

In another aspect, the invention provides a method for to preventing,inhibiting or treating neuropathic pain, wherein the method comprisesadministering an effective amount of at least one compound of formula Ior a compound of formula I and a pharmaceutically-acceptable carrier isadministered to a subject in need thereof. Pain can be nociceptive orneuropathic in nature. Neuropathic pain is characterized by its chronicnature, an absence of an obvious, direct cause (e.g., tissue damage),hyperalgesia or allodynia. Hyperalgesia is an exaggerated response to apainful stimulus. Allodynia is the perception of normal stimuli aspainful (examples include the touch of clothing, warm or cool air,etc.). Neuropathic pain can be a sequel to nerve damage in an extremitysuch as an arm, or more often a leg. Precipitating events can includetrauma, e.g., motor vehicle accidents or amputations (e.g., phantom limbpain). Neuropathic pain can occur due to an adverse effect of drugtherapies, e.g., vincristine or paclitaxel (TAXOL™) or can occur as acomponent of disease pathologies, such as diabetes type 1 or type2,shingles, HIV-1 infections, etc. Typically, neuropathic pain is notresponsive to opiates or non-steroidal anti-inflammatory drugs such asaspirin.

In another aspect, the invention provides a method for repairingvascular injury following catheterization, comprising contacting thelumen of the affected vessel with an effective amount of the compound offormula I. In another aspect, the invention includes coating indwellingstents with a compound of formula I.

In another aspect, the present invention provides compositions andmethods for the use of S1P analogs to prevent and inhibit vascularrestenosis following vascular injury. For example, the injury can be dueto balloon angioplasty. In another aspect, the present inventionincludes a method for treating subjects to prevent vascular restenosis.

In another aspect, the present invention provides compositions andmethods for the use of sphingosine analogs (including S1P pro-drugs) toprevent asthma attacks. In one aspect, the asthma could be due to overproduction of cysteinyl leukotrienes. In another aspect, the presentinvention includes a method for treating subjects to treat asthma.

In another aspect, the present invention provides compositions andmethods for the use of sphingosine analogs of formula I (including S1Ppro-drugs) to treat obesity.

In another aspect, the present invention provides compositions andmethods for the use of sphingosine analogs (including S1P pro-drugs) tonormalize blood lipid composition. In one aspect, blood low densitylipoprotein (LDL or ‘bad cholesterol’) levels could be lowered. Inanother aspect, blood triglyceride levels could be lowered.

In another aspect, the present invention provides compositions andmethods for the use of S1P analogs and S1P pro-drugs for the preventionand treatment of arteriosclerosis.

In another aspect, the present invention provides compositions andmethods for the use of S1P analogs and S1P pro-drugs for the treatmentof neoplastic disease. In one aspect, this treatment is effected byapplication of S1P receptor antagonists that are efficacious by virtueof their anti-angiogenic properties. In another aspect, the treatment iseffected by administration of sphingosine analogs of formula I thatinhibit the multiple substrate lipid kinase.

In another aspect, the present invention provides compositions andmethods for the use of S1P analogs and S1P pro-drugs for the treatmentof neurodegenerative diseases. In one aspect, the treatment is forsenile dementia of the Alzheimers type.

In another aspect, the invention provides a compound of formula I, or apharmaceutically acceptable salt thereof for use in medical treatment(for example, treatment of neoplastic disease, treatment of neuropathicpain, treatment of autoimmune disease, prolongation of allograftsurvival).

In another aspect, the invention provides a method for the use of acompound of formula I or a pharmaceutically acceptable salt thereof toprepare a medicament for inhibiting tumor growth, metastasis or tumorangiogenesis in a mammalian species (for example, a human).

In another aspect, the invention provides for the use of a compound offormula I or a pharmaceutically acceptable salt thereof to prepare amedicament for treating an autoimmune disease or prolonging allograftsurvival in a mammalian species (for example, a human).

In another aspect, the invention provides for the use of a compound offormula I or a pharmaceutically acceptable salt thereof to prepare amedicament for treating neuropathic pain in a mammalian species (forexample, a human).

In another aspect, the invention provides a method for assessing acompound of formula I (e.g., S1P receptor pro-drugs) as a substrate forsphingosine kinase types 1 or 2, in vitro and in vivo. In anotheraspect, the invention includes a method of assessing a compound offormula I for binding to designated receptor sites comprising in vivo orin vitro, with an amount of a compound of formula I effective to bindsaid receptors. Tissue comprising ligand bound designated S1P receptorsites can be used to measure the selectivity of test compounds forspecific receptor subtypes, or can be used as a tool to identifypotential therapeutic agents for the treatment of diseases, bycontacting said agents with said ligand-receptor complexes, andmeasuring the extent of displacement of the ligand or binding of theagent.

In another aspect, the invention provides novel intermediates andprocesses disclosed herein that are useful for preparing compounds offormula I, including the generic and specific intermediates as well asthe synthetic processes described herein.

In another aspect, the present invention provides synthetic schemes andmethods of use of compounds having formula I and analogs or derivativesthereof. In another aspect, the invention provides synthetic andmodification schemes for preparing analogs and derivatives of thecompounds of formula I, as well as compositions and methods for the useof such analogs and derivatives.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Other features, objects, andadvantages of the invention will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of three S1P agonists FTY720, AA151 compoundXXIX and additional compounds of formula I.

FIGS. 2, 3 and 4 are schemes illustrating syntheses of compounds offormula I.

FIGS. 5A, 5B and 6 illustrate the results of sphingosine kinase type 2(SPHK2) activity for S1P agonist compounds FTY720, AA151, VIII andXVIII.

FIGS. 7 and 8 graphically illustrate the results of a broken cellGTPγ³⁵S binding assay for the human S1P₁ receptor, testing S1P andcompounds XXVI, XXVII and XXVIII.

FIG. 9 is graphically illustrates total lymphocyte counts (k/μl) 24hours (left bar of each group) and 48 hours (right bar of each group)after an oral dose of the test compounds are administered to mice.

FIG. 10 is graphically illustrates total lymphocyte counts (k/μl) 18hours after an oral dose of the test compounds are administered to mice.

FIG. 11 is a graphical representation of the results of an assayillustrating that compound VIII has no effect on the heart rate of mice.In the assay the test compound was administered via IV Dosing and thevehicle was 2% cyclodextrin.

DETAILED DESCRIPTION

The following abbreviations are used herein: S1P,sphingosine-1-phosphate; S1P₁₋₅ S1P receptor types; GPCR, G-proteincoupled receptor; SAR, structure-activity relationship; EDG, endothelialcell differentiation gene; EAE, experimental autoimmuneencephalomyelitis; NOD non-obese diabetic; TNFα, tumor necrosis factoralpha; HDL, high density lipoprotein; and RT-PCR, reverse transcriptasepolymerase chain reaction.

In describing and claiming the invention, unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Although any materials and methods similar orequivalent to those described herein can be used in the practice ortesting of the present invention, the preferred materials and methodsare described herein. Each of the following terms has meaning associatedwith it in this section. Specific and preferred values listed below forradicals, substituents, and ranges are for illustrations only; they donot exclude other defined values or other values within defined rangesfor the radicals and substituents.

The terms “a,” “an,” “the,” “at least one,” and “one or more” are usedinterchangeably. Thus, for example, a composition that comprises “an”element means one element or more than one element.

The term “receptor agonists” are compounds that mimic the action of S1Pat one or more of its receptors but may have differing potency orefficacy. The term “receptor antagonists” are compounds that 1) lackintrinsic agonist activity and 2) block agonist (e.g., S1P) activationof the S1P receptor(s), often in a manner that is both fullysurmountable and reversible (‘competitive antagonist’).

The term “affected cell” refers to a cell of a subject afflicted with adisease or disorder, which affected cell has an altered phenotyperelative to a subject not afflicted with a disease or disorder.

Cells or tissue are “affected” by a disease or disorder if the cells ortissue have an altered phenotype relative to the same cells or tissue ina subject not afflicted with a disease or disorder.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, is reduced.

An “analog” of a chemical compound is a compound that, by way ofexample, resembles another in structure but is not necessarily an isomer(e.g., 5-fluorouracil is an analog of thymine).

The terms “cell,” “cell line,” and “cell culture” may be usedinterchangeably.

A “control” cell, tissue, sample, or subject is a cell, tissue, sample,or subject of the same type as a test cell, tissue, sample, or subject.The control may, for example, be examined at precisely or nearly thesame time the test cell, tissue, sample, or subject is examined. Thecontrol may also, for example, be examined at a time distant from thetime at which the test cell, tissue, sample, or subject is examined, andthe results of the examination of the control may be recorded so thatthe recorded results may be compared with results obtained byexamination of a test cell, tissue, sample, or subject. The control mayalso be obtained from another source or similar source other than thetest group or a test subject, where the test sample is obtained from asubject suspected of having a disease or disorder for which the test isbeing performed.

A “test” cell, tissue, sample, or subject is one being examined ortreated.

A “pathoindicative” cell, tissue, or sample is one which, when present,is an indication that the animal in which the cell, tissue, or sample islocated (or from which the tissue was obtained) is afflicted with adisease or disorder. By way of example, the presence of one or morebreast cells in a lung tissue of an animal is an indication that theanimal is afflicted with metastatic breast cancer.

A tissue “normally comprises” a cell if one or more of the cell arepresent in the tissue in an animal not afflicted with a disease ordisorder.

The use of the word “detect” and its grammatical variants is meant torefer to measurement of the species without quantification, whereas useof the word “determine” or “measure” with their grammatical variants aremeant to refer to measurement of the species with quantification. Theterms “detect” and “identify” are used interchangeably herein.

A “detectable marker” or a “reporter molecule” is an atom or a moleculethat permits the specific detection of a compound comprising the markerin the presence of similar compounds without a marker. Detectablemarkers or reporter molecules include, e.g., radioactive isotopes,antigenic determinants, enzymes, nucleic acids available forhybridization, chromophores, fluorophores, chemiluminescent molecules,electrochemically detectable molecules, and molecules that provide foraltered fluorescence-polarization or altered light-scattering.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

A “disorder” in an animal is a state of health in which the animal isable to maintain homeostasis, but in which the animal's state of healthis less favorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” means an amount sufficient to produce a selectedeffect. For example, an effective amount of an S1P receptor antagonistis an amount that decreases the cell signaling activity of the S1Preceptor.

A “functional” molecule is a molecule in a form in which it exhibits aproperty by which it is characterized. By way of example, a functionalenzyme is one which exhibits the characteristic catalytic activity bywhich the enzyme is characterized.

The term “inhibit” refers to the ability of a disclosed compound toreduce or impede a described function. Preferably, inhibition is by atleast 10%, more preferably by at least 25%, even more preferably by atleast 50%, and most preferably, the function is inhibited by at least75%.

“Instructional material” includes a publication, a recording, a diagram,or any other medium of expression which can be used to communicate theusefulness of the disclosed compounds in the kit for effectingalleviation of the various diseases or disorders recited herein.Optionally, or alternately, the instructional material may describe oneor more methods of alleviating the diseases or disorders in a cell or atissue of a mammal. The instructional material of the kit may, forexample, be affixed to a container which contains a disclosed compoundor be shipped together with a container which contains the identifiedcompound. Alternatively, the instructional material may be shippedseparately from the container with the intention that the instructionalmaterial and the compound be used cooperatively by the recipient.

The term “parenteral” means not through the alimentary canal but by someother route such as subcutaneous, intramuscular, intraspinal, orintravenous.

The term “pharmaceutically acceptable carrier” includes any of thestandard pharmaceutical carriers, such as a phosphate buffered salinesolution, water and emulsions such as an oil/water or water/oilemulsion, and various types of wetting agents. The term also encompassesany of the agents approved by a regulatory agency of the U.S. Federalgovernment or listed in the U.S. Pharmacopeia for use in animals,including humans.

The term “purified” and similar terms relate to the isolation of amolecule or compound in a form that is substantially free (at least 75%free, preferably 90% free, and most preferably at least 95% free) fromother components normally associated with the molecule or compound in anative environment. The term “purified” does not necessarily indicatethat complete purity of the particular molecules achieved during theprocess. A “very pure” compound refers to a compound that is greaterthan 90% pure. A “highly purified” compound refers to a compound that isgreater than 95% pure.

A “sample” refers preferably to a biological sample from a subject,including, but not limited to, normal tissue samples, diseased tissuesamples, biopsies, blood, saliva, feces, semen, tears, and urine. Asample can also be any other source of material obtained from a subject,which contains cells, tissues, or fluid of interest. A sample can alsobe obtained from cell or tissue culture.

The term “standard,” refers to something used for comparison. Forexample, a standard can be a known standard agent or compound which isadministered or added to a control sample and used for comparing resultswhen measuring said compound in a test sample. Standard can also referto an “internal standard,” such as an agent or compound which is addedat known amounts to a sample and is useful in determining such things aspurification or recovery rates when a sample is processed or subjectedto purification or extraction procedures before a marker of interest ismeasured.

A “subject” of analysis, diagnosis, or treatment is an animal. Suchanimals include mammals, preferably a human.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

A “therapeutically effective amount” of a compound is that amount ofcompound which is sufficient to provide a beneficial effect to thesubject to which the compound is administered.

The term “treating” includes prophylaxis of the specific disorder orcondition, or alleviation of the symptoms associated with a specificdisorder or condition or preventing or eliminating said symptoms.

The disclosed compounds are generally named according to the IUPAC orCAS nomenclature system. Abbreviations which are well known to one ofordinary skill in the art may be used (e.g., “Ph” for phenyl, “Me” formethyl, “Et” for ethyl, “h” for hour or hours, “rt” for roomtemperature, and “rac” for racemic mixture).

The values listed below for radicals, substituents, and ranges, are forillustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents. Thedisclosed compounds include compounds of formula I having anycombination of the values, specific values, more specific values, andpreferred values described herein.

The term “halogen” or “halo” includes bromo, chloro, fluoro, and iodo.The term “haloalkyl”, refers to an alkyl radical bearing at least onehalogen substituent, non-limiting examples include, but are not limitedto, chloromethyl, fluoroethyl or trifluoromethyl and the like. The term“C₁-C₂₀ alkyl” refers to a branched or linear alkyl group having fromone to nine carbons. Non-limiting examples include, but are not limitedto, methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl,tert-butyl, pentyl, hexyl, heptyl, octyl and the like. The term “C₂-C₂₀alkenyl”, refers to an olefinically unsaturated branched or linear grouphaving from two to nine carbon atoms and at least one double bond.Typically, C₂-C₂₀ alkenyl groups include, but are not limited to,1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl,hexenyl, heptenyl, octenyl and the like. The term (C₂-C₂₀)alkynyl can beethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl,1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl,3-hexynyl, 4-hexynyl, or 5-hexynyl, and the like. The term“(C₁-C₁₀)alkoxy or alkoxyl” refers to an alkyl group attached through anoxygen atom. Examples of (C₁-C₁₀)alkoxy can be methoxy, ethoxy, propoxy,isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, orhexyloxy and the like. The term “C₃-C₁₂ cycloalkyl”, can be cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and thelike.

The term “optionally substituted” refers to zero, one, two, three orfour substituents, wherein the substituents are each independentlyselected. Each of the independently selected substituents may be thesame or different than other substituents.

The term “(C₆-C₁₀)aryl” refers to a mono or bicyclic carbocyclic ringsystem having one or two aromatic rings including, but not limited to,phenyl, benzyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and thelike.

The term “aryl(C₁-C₂₀)alkyl” or “aralkyl” refers to an alkyl groupsubstituted with a mono or bicyclic carbocyclic ring system having oneor two aromatic rings including, a group such as phenyl, naphthyl,tetrahydronaphthyl, indanyl, indenyl, and the like. Non-limitingexamples of arylalkyl include benzyl, phenylethyl, and the like.

The term “optionally substituted aryl” includes aryl compounds havingzero, one, two, three or four substituents, and a substituted arylincludes aryl compounds having one, two, three or four substituents,wherein the substituents include groups such as, for example, alkyl,halo, or amino substituents.

The “(C₂-C₁₀)heterocyclic group” refers to an optionally substitutedmono- or bicyclic carbocyclic ring system containing one, two, or threeheteroatoms (optionally in each ring) wherein the heteroatoms areoxygen, sulfur, and nitrogen.

The term “(C₄-C₁₀)heteroaryl” refers to an optionally substituted mono-or bicyclic carbocyclic ring system containing one, two, or threeheteroatoms (optionally in each ring) wherein the heteroatoms areoxygen, sulfur, and nitrogen. Non-limiting examples of heteroaryl groupsinclude furyl, thienyl, pyridyl, and the like.

The term “bicyclic” represents either an unsaturated or saturated stablebridged or fused bicyclic carbon ring. The bicyclic ring may be attachedat any carbon atom which affords a stable structure. Typically abicyclic ring system can have from about 7- to about 12 atoms in thering system. The term includes, but is not limited to, naphthyl,dicyclohexyl, dicyclohexenyl, and the like.

The term “phosphate analog” and “phosphonate analog” comprise analogs ofphosphate and phosphonate wherein the phosphorous atom is in the +5oxidation state and one or more of the oxygen atoms is replaced with anon-oxygen moiety, including for example, the phosphate analogsphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate,phosphoramidate, boronophosphates, and the like, including associatedcounterions, e.g., H, NH₄, Na, K, and the like if such counterions arepresent.

A “derivative” of a compound refers to a chemical compound that may beproduced from another compound of similar structure in one or moresteps, such as replacement of hydrogen by an alkyl, acyl, or aminogroup.

The term “pharmaceutically acceptable carrier” includes any of thestandard pharmaceutical carriers, such as a phosphate buffered salinesolution, hydroxypropyl beta-cyclodextrins (HO-propyl betacyclodextrins), water, emulsions such as an oil/water or water/oilemulsion, and various types of wetting agents. The term also encompassesany of the agents approved by a regulatory agency of the U.S. Federalgovernment or listed in the U.S. Pharmacopeia for use in animals,including humans.

The term “pharmaceutically-acceptable salt” refers to salts which retainthe biological effectiveness and properties of the disclosed compoundsand which are not biologically or otherwise undesirable. In many cases,the disclosed compounds are capable of forming acid or base salts byvirtue of the presence of amino or carboxyl groups or groups similarthereto.

An “effective amount” means an amount sufficient to produce a selectedeffect. For example, an effective amount of an S1P receptor agonist isan amount that decreases the cell signaling activity of the S1Preceptor.

The disclosed compounds can contain one or more asymmetric centers inthe molecule. In accordance with the present disclosure any structurethat does not designate the stereochemistry is to be understood asembracing all the various optical isomers, as well as racemic mixturesthereof.

The disclosed compounds may exist in tautomeric forms and the inventionincludes both mixtures and separate individual tautomers. For example,the following structure:

is understood to represent a mixture of the structures:

as well as

The terms 16:0, 18:0, 18:1, 20:4 or 22:6 hydrocarbon refers to abranched or straight alkyl or alkenyl group, wherein the first integerrepresents the total number of carbons in the group and the secondinteger represent the number of double bonds in the group.

An “S1P modulating agent” refers a compound or composition that iscapable of inducing a detectable change in S1P receptor activity in vivoor in vitro (e.g., at least 10% increase or decrease in S1P activity asmeasured by a given assay such as the bioassay described in the examplesand known in the art. “S1P receptor,” refers to all of the S1P receptorsubtypes (for example, the S1P receptors S1P₁, S1P₂, S1P₃, S1P₄, andS1P₅), unless the specific subtype is indicated.

It will be appreciated by those skilled in the art that the disclosedcompounds having chiral centers may exist in and be isolated inoptically active and racemic forms. It is to be understood that thedisclosed compounds encompass any racemic, optically active orstereoisomeric form, or mixtures thereof, of the compound, which possessthe useful properties described herein, such as the S,R; S,S; R,R; orR,S diastereomers. It is well known in the art how to prepare suchoptically active forms (for example, by resolution of the racemic formby recrystallization techniques, by synthesis from optically-activestarting materials, by chiral synthesis, or by chromatographicseparation using a chiral stationary phase) and how to determine S1Pagonist activity using the standard tests described herein, or usingother similar tests which are well known in the art. In addition, somecompounds may exhibit polymorphism.

Potential uses of an S1P receptor agonist pro-drugs (S1P₁ receptor typeselective agonists preferred) include, but are not limited to, alteringlymphocyte trafficking as a method of treatment for autoimmunepathologies such as uveitis, type I diabetes, rheumatoid arthritis,inflammatory bowel diseases, and, most particularly, multiple sclerosis.“Treatment” of multiple sclerosis includes the various forms of thedisease including relapsing-remitting, chronic progressive, etc., andthe S1P receptor agonists can be used alone or in conjunction with otheragents to relieve signs and symptoms of the disease as well asprophylactically.

In addition, the disclosed compounds can be used for altering lymphocytetrafficking as a method for prolonging allograft survival, for examplesolid organ transplants, treatment of graft vs. host disease, bonemarrow transplantation, and the like.

In addition, the disclosed compounds can be used to inhibit autotaxin.Autotaxin, a plasma phosphodiesterase, has been demonstrated to undergoend product inhibition. Autotaxin hydrolyzes several substrates to yieldlysophosphatidic acid and sphingosine 1-phosphate, and has beenimplicated in cancer progression and angiogenesis. Therefore, S1Preceptor agonist pro-drugs of the disclosed compounds can be used toinhibit autotaxin. This activity may be combined with agonism at S1Preceptors or may be independent of such activity.

In addition, disclosed compounds can be useful for inhibition of S1Plyase. S1P lyase is an intracellular enzyme that irreversibly degradesS1P. Inhibition of S1P lyase disrupts lymphocyte trafficking withconcomitant lymphopenia. Accordingly, S1P lyase inhibitors can be usefulin modulating immune system function. Therefore, the disclosed compoundscan be used to inhibit S1P lyase. This inhibition could be in concertwith S1P receptor activity, or be independent of activity at any S1Preceptor.

In addition, disclosed compounds can be useful as antagonists of thecannabinoid CB₁ receptor. CB₁ antagonism is associated with a decreasein body weight and an improvement in blood lipid profiles. The CB₁antagonism could be in concert with S1P receptor activity, or beindependent of activity at any S1P receptor.

In addition, disclosed compounds can be useful for inhibition of groupIVA cytosolic PLA₂ (cPLA₂). cPLA₂ catalyzes the release of eicosanoicacids (e.g., arachidonic acid). The eicosanoic acids are transformed topro-inflammatory eicosanoids such as prostaglandins and leukotrienes.Thus, disclosed compounds may be useful as anti-inflammatory agents.This inhibition could be in concert with S1P receptor activity, or beindependent of activity at any S1P receptor.

In addition, disclosed compounds may be useful for inhibition of themultiple substrate lipid kinase (MuLK). MuLK is highly expressed in manyhuman tumor cells and thus its inhibition might slow the growth orspread of tumors.

“Treatment” of multiple sclerosis includes the various forms of thedisease including relapsing-remitting, chronic progressive, etc., andthe S1P receptor agonists can be used alone or in conjunction with otheragents to relieve signs and symptoms of the disease as well asprophylactically.

The present invention is also includes pharmaceutical compositionscomprising the compounds of formula I. More particularly, such compoundscan be formulated as pharmaceutical compositions using standardpharmaceutically acceptable carriers, fillers, solubilizing agents andstabilizers known to those skilled in the art. For example, apharmaceutical composition comprising a compound of formula I, oranalog, derivative, or modification thereof, as described herein, isused to administer the appropriate compound to a subject.

The compounds of formula I are useful for treating a disease or disorderincluding administering to a subject in need thereof of atherapeutically acceptable amount of a compound of formula I, or apharmaceutical composition comprising a therapeutically effective amountof a compound of formula I, and a pharmaceutically-acceptable carrier.

The disclosed compounds and method are directed to sphingosine1-phosphate (S1P) analogs that have activity as receptor receptoragonists or antagonists at one or more S1P receptors, specifically theS1P₁, S1P₄ and S1P₅ receptor types. The disclosed compounds and methodinclude both compounds that have a phosphate moiety as well as compoundswith hydrolysis-resistant phosphate surrogates such as phosphonates,alpha-substituted phosphonates particularly where the alpha substitutionis a halogen and phosphothionates.

The values listed below for radicals, substituents, and ranges, are forillustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents.

X¹, Y¹ and Z¹ are independently O, CH, CH₂, CHCF₃, N, NH, or S.

Another value for X¹, Y¹ and Z¹ is CH₂.

R¹ can be hydrogen, fluorine, chlorine, bromine, trifluoromethyl,methoxy, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, or (C₁-C₆)alkyl substitutedwith, alkoxy or cyano.

Additional values for R¹ are hydrogen, trifluoromethyl, or —CH₂CF₃.

More additional values for R¹ are alkyl-substituted aryl,aryl-substituted alkyl, or aryl-substituted arylalkyl.

More additional values for R¹ are benzyl, phenylethyl, or methyl benzyl.

Compounds having formula I can have an R² group that includes a chainhaving the structure —CH₂—CH₂—O—CH₂—CH₂—O—.

Values for R² include

Additional values for R² having formula II are

where Y³ is (CH₃)₃C—, CH₃CH₂(CH₃)₂C—, CH₃CH₂CH₂—, CH₃(CH₂)₂CH₂—,CH₃(CH₂)₄—CH₂—(CH₃)₂CHCH₂—, (CH₃)₃CCH₂—, CH₃CH₂O—, (CH₃)₂CHO—, orCF₃CH₂CH₂— or a group having the formula:

An additional value for R² having formula II (para substituted3,5-diphenyl-(1,2,4)-oxadiazole) is;

Another value for R² having formula II is;

Another value for R² having formula II is;

Additional values for R² having formula III are;

Another value for R² having formula III is;

Additional values for R² include (C₁-C₂₀)alkyl, (C₁-C₂₀)alkoxy, or(C₂-C₂₆)alkoxyalkyl.

More additional values for R² include (C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl and(C₂-C₁₄)alkynyl or (C₁-C₁₀)alkoxy optionally substituted with carbonyl(C═O) or oxime (C═NR^(d)) groups.

Additional values for R² include methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, trifluoromethyl, trifluoroethyl, trifluoromethoxy,trifluoroethoxy, methoxy, ethoxy, propoxy, butoxy, pentoxy, heptoxy, oroctoxy.

Another value for R³ is methyl, hydroxymethyl, ethyl, hydroxyethyl,propyl, hydroxypropyl, or isopropyl.

Another value for R³ is methyl, hydroxymethyl, ethyl, or hydroxyethyl.

A value for R⁴ is hydroxy, or phosphate (—OPO₃H₂).

A specific compound has the formula

Additional compounds have formulas

Additional compounds having formula I include compounds above or in FIG.1 where one of more of the hydrogen atoms from a hydroxy group isreplaced with a phosphate group —OP(═O)(OH)₂.

Additional compounds of formula I are illustrated in table 1, below.

TABLE 1

Compound R^(e) XX

XXI

XXII

XXIII

XXIV

XXV

In another aspect, the invention provides S1P receptor pro-drugcompounds having the general structure of formula I, is provided by acompound with a mono-substituted tetralin ring system that has theformula VIII. In some embodiments of formula I, the structure (e.g., IXand X) has only a single chiral center and that the amino carbon ispro-chiral, e.g., will become chiral following enzyme-catalyzedphosphorylation.

Without wishing to be bound by any particular theory, it is expectedthat the compounds described herein are pro-drugs, e.g., are activatedby phosphorylation of the primary alcohol to form themono-phosphorylated analog. Additionally, the active drugs are expectedto be agonists at the S1P type 1 receptor.

In cases where compounds of formula I are sufficiently basic or acidicto form stable nontoxic acid or base salts, preparation andadministration of the compounds as pharmaceutically acceptable salts maybe appropriate. Examples of pharmaceutically acceptable salts areorganic acid addition salts formed with acids which form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartarate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Inorganic salts may also beformed, including hydrochloride, sulfate, nitrate, bicarbonate, andcarbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts from inorganic bases, include but arenot limited to, sodium, potassium, lithium, ammonium, calcium andmagnesium salts. Salts derived from organic bases include, but are notlimited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri(substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,heteroaryl, or heterocyclic and the like. Also included are amines wherethe two or three substituents, together with the amino nitrogen, form aheterocyclic or heteroaryl group. Mon-limiting examples of aminesinclude, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine,morpholine, N-ethylpiperidine, and the like. It should also beunderstood that other carboxylic acid derivatives would be useful, forexample, carboxylic acid amides, including carboxamides, lower alkylcarboxamides, dialkyl carboxamides, and the like.

The compounds of formula I can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of forms adapted to the chosen route ofadministration, e.g., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least about 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

Exemplary pharmaceutical dosage forms for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, e.g., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Exemplary solid carriers include finely divided solids such as talc,clay, microcrystalline cellulose, silica, alumina and the like. Usefulliquid carriers include water, alcohols or glycols orwater-alcohol/glycol blends, in which the present compounds can bedissolved or dispersed at effective levels, optionally with the aid ofnon-toxic surfactants. Adjuvants such as fragrances and additionalantimicrobial agents can be added to optimize the properties for a givenuse. The resultant liquid compositions can be applied from absorbentpads, used to impregnate bandages and other dressings, or sprayed ontothe affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of formula I to the skin are known to the art; forexample, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

Generally, the concentration of the compound(s) of formula I in a liquidcomposition, such as a lotion, will be from about 0.1 to about 25 weightpercent, preferably from about 0.5-10 weight percent. The concentrationin a semi-solid or solid composition such as a gel or a powder will beabout 0.1-5 wt-%, preferably about 0.5-2.5 weight percent based on thetotal weight of the composition.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician. In general, however, a dose will be in the range of fromabout 0.1 to about 10 mg/kg of body weight per day.

The compound is conveniently administered in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.5 to about75 μM, preferably, about 1 to 50 μM, most preferably, about 2 to about30 μM. This may be achieved, for example, by the intravenous injectionof a 0.05 to 5% solution of the active ingredient, optionally in saline,or orally administered as a bolus containing about 1-100 mg of theactive ingredient. Desirable blood levels may be maintained bycontinuous infusion to provide about 0.01-5.0 mg/kg/hr or byintermittent infusions containing about 0.4-15 mg/kg of the activeingredient(s).

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four, or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

The disclosed method includes a kit comprising an inhibitor compound offormula I and instructional material which describes administering theinhibitor compound or a composition comprising the inhibitor compound toa cell or a subject. This should be construed to include otherembodiments of kits that are known to those skilled in the art, such asa kit comprising a (preferably sterile) solvent for dissolving orsuspending the inhibitor compound or composition prior to administeringthe compound or composition to a cell or a subject. Preferably, thesubject is a human.

In accordance with the disclosed compounds and methods, as describedabove or as discussed in the Examples below, there can be employedconventional chemical, cellular, histochemical, biochemical, molecularbiology, microbiology, and in vivo techniques which are known to thoseof skill in the art. Such techniques are explained fully in theliterature.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the disclosed compounds.

Processes for preparing compounds of formula I or for preparingintermediates useful for preparing compounds of formula I are providedas further embodiments. Intermediates useful for preparing compounds offormula I are also provided as further embodiments. The processes areprovided as further embodiments and are illustrated in the schemesherein wherein the meanings of the generic radicals are as given aboveunless otherwise qualified.

An example of the synthesis of several disclosed compounds isillustrated in Scheme 1 (FIG. 2). The reagents and conditions are asfollows: a) Tf₂O, 2,6-lutidine, CH₂Cl₂, 0° C., 2 h, 93%; b) 1-octene,9-BBN, K₃PO₄, KBr, H₂O, Pd(PPh₃)₄, 65° C., 2 h, 82%; c) CuBr₂, EtOAc,CHCl₃, reflux 6 h, 80%; d) NaH, N-acetamido-diethylmalonate, DMF, 0°C.-rt, overnight, 75%; e) Et₃SH, TiCl₄,CH₂Cl₂, rt, 12 h, 65%; f) LiBH₄,rt, THF, 48 h, 33%; g) LiOH, H₂O, MeOH, THF, 50° C., 5 h, 75%; h) P₂O₅,H₃PO₄, 100° C., 2 h, 37%; i) 12M HCL, MeOH, reflux, 2 h; j) LiAlH₄, THF,reflux, 12 h, 21%, two steps; k) P₂O₅, H₃PO₄, 100° C., 2 h, 50%.

The invention is now described with reference to the following Examplesand Embodiments. Without further description, it is believed that one ofordinary skill in the art can, using the preceding description and thefollowing illustrative examples, make and utilize the disclosedcompounds. The following working examples therefore, are provided forthe purpose of illustration only and specifically point out thepreferred embodiments, and are not to be construed as limiting in anyway the remainder of the disclosure. Therefore, the examples should beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

EXAMPLES Example 1 Trifluoromethanesulfonic acid5-oxo-5,6,7,8-tetrahydro-naphthalen-2-yl ester (2)

Trifluoromethanesulfonic anhydride (1.7 mL, 10 mmol) was added slowlyover 1 hour to a solution of 6-hydroxy-1-tetralone (1.62 g, 10 mmol) and2,6-lutidine (1.28 mL, 10 mmol) in dry dichloromethane (10 mL) cooled to0° C. After 1 hour the solution was diluted with dichloromethane (10 mL)and washed with 1 M hydrochloric acid (20 mL). The organic layer wasre-extracted with dichloromethane (50 mL) and the combined organicswashed with 1 M hydrochloric acid (10 mL). The organics were dried overmagnesium sulfate and concentrated under vacuum. The residue waspurified by column chromatography (Silica gel, CH₂Cl₂) to provide 2.7 gof compound 2 (93%).

¹H NMR (300 MHz, CDCl₃) δ 2.13 (p, 2H, J=6.22 Hz), 2.63 (t, 2H, J=6.95Hz), 2.98 (t, 2H, J=6.22 Hz), 7.15 (m, 2H), 8.07 (m, 1H); ¹³C NMR δ23.08, 29.88, 38.92, 116.74, 119.81, 121.56, 130.14, 132.58, 147.38,152.52, 196.53.

Example 2 6-Octyl-3,4-dihydro-2H-naphthalen-1-one (3)

9-BBN (0.5 M solution in THF, 20.2 mL, 10.1 mmol) was added to 1-octene(1.6 mL, 10.1 mmol) at room temperature. The solution was stirred, atroom temperature, overnight. After this time, K₃PO₄ (2.93 g, 13.8 mmol),Pd(Ph₃P)₄ (191 mg, 0.17 mmol, 1.8 mol %), KBr (1.2 g, 10.1 mmol) anddegassed H₂O (0.18 mL, 10 mmol) were added. This was followed by asolution of compound 2 (2.7 g, 9.2 mmol) in dry degassed THF (10 mL).The reaction mixture was heated at 65° C. under argon for 2 hours. Aftercooling, the solution was acidified to pH 1 and extracted into EtOAc(100 mL). The aqueous layer was re-extracted with EtOAc (50 mL) and thecombined organics washed with H₂O (20 mL) and brine (40 mL). The organiclayer was dried over magnesium sulfate and concentrated under vacuum.The residue was purified by column chromatography (Silica gel, 5% EtOAcin hexanes) to provide 1.93 g of compound 3 (82%).

¹H NMR (300 MHz, CDCl₃) δ 0.85 (t, 3H, J=6.95 Hz), 1.24 (bs, 10H), 1.58(p, 2H, J=6.95 Hz), 2.06 (p, 2H, J=5.85 Hz), 2.57 (t, 4H, J=6.95 Hz),2.87 (t, 2H, J=6.22 Hz), 7.01 (s, 1H), 7.06 (d, 1H, J=8.05 Hz), 7.91 (d,1H, J=8.06 Hz); ¹³C NMR δ 14.32, 22.88, 23.61, 29.44, 29.55, 29.66,29.96, 31.32, 32.08, 36.31, 39.33, 127.12, 127.45, 128.73, 130.75,144.70, 149.28, 198.09.

Example 3 2-Bromo-6-octyl-3,4-dihydro-2H-naphthalen-1-one (4)

Cupric bromide (3.34 g, 15.0 mmol,) was heated at reflux in ethylacetate (10 mL) with stirring. To this was added compound 3 (1.93 g, 7.5mmol) in chloroform (10 mL). The reaction was heated at reflux for afurther 6 hours and cooled. Copper bromide and cupric bromide residueswere filtered off and the filtrate was decolorized with activatedcharcoal and filtered through a bed of Celite and washed with ethylacetate (4×50 mL). The solvent was removed under reduced pressure andthe residue was purified by column chromatography (Silica gel, 2% EtOAcin hexanes) to provide 2.02 g of compound 4 (80%).

¹H NMR (300 MHz, CDCl₃) δ 0.87 (t, 3H, J=6.95 Hz), 1.26 (bs, 10H), 1.61(p, 2H, J=6.96 Hz), 2.46 (m, 2H), 2.62 (t, 2H, J=7.69 Hz), 2.86 (dt, 1H,J=16.34 Hz, 4.39 Hz), 3.27 (dt, 1H, J=16.83 Hz, 4.39 Hz), 4.69 (t, 1H,J=4.02 Hz), 7.07 (s, 1H), 7.14 (d, 1H, J=8.05 Hz), 7.99 (d, 1H, J=8.05Hz); ¹³C NMR δ 14.34, 22.88, 26.42, 29.44, 29.57, 29.64, 31.25, 32.08,32.32, 36.39, 127.75, 128.00, 128.73, 129.00, 144.30, 150.39, 190.54.

Example 42-Acetylamino-2-(6-octyl-1-oxo-1,2,3,4-tetrahydro-naphthalen-2-yl)-malonicacid diethyl ester (5)

Sodium hydride (720 mg, 18.0 mmol) 60% in mineral oil was suspended indry DMF (10 mL) and a solution of diethyl acetamidomalonate (3.26 g, 15mmol) in dry DMF (10 mL) was added. The solution was stirred at 0° C.for 3 hours until the anion had formed. A solution of 4 (2.02 g, 6.0mmol) in dry DMF (10 mL) was added and the solution warmed to roomtemperature and stirred overnight. The mixture was poured into distilledwater (50 mL), in an ice-bath, acidified to pH 3 with 1M hydrochloricacid and extracted with ethyl acetate (3×50 mL). The organic phases werewashed with brine (2×30 mL) and dried over magnesium sulfate andconcentrated under vacuum. The residue was purified by columnchromatography (Silica gel, 40% EtOAc in hexanes) to provide 2.12 g ofcompound 5 (75%).

¹H NMR (300 MHz, CDCl₃) δ 0.85 (t, 3H, J=6.22 Hz), 1.24 (m, 16H), 1.58(p, 2H, J=6.95 Hz), 1.97 (s, 3H), 2.59 (t, 2H, J=7.32 Hz), 2.83-3.21 (m,4H), 3.88 (dd, 1H, J=14.00 Hz, 3.68 Hz), 4.14-4.32 (m, 4H), 6.86 (s,1H), 7.03 (s, 1H), 7.07 (d, 1H, J=8.69 Hz), 7.84 (d, 1H, J=8.36 Hz); ¹³CNMR δ 14.05, 14.16, 14.30, 22.85, 23.31, 26.98, 29.40, 29.49, 29.61,29.98, 31.28, 32.05, 36.32, 56.16, 62.40, 63.13, 66.33, 127.16, 127.63,128.78, 144.84, 150.07, 166.38, 168.70, 169.83, 197.63.

Example 52-Acetylamino-2-(6-octyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-malonicacid diethyl ester (6)

To a solution of triethylsilane (1.3 ml, 8.2 mmol) in 5 ml of CH₂Cl₂ wasadded compound 5 (1 g, 2.1 mmol) in 5 ml of CH₂Cl₂. The reaction mixturewas stirred at room temperature under Ar and TiCl₄ (0.09 ml, 8.2 mmol)was added dropwise. The resulting solution was stirred for 12 hours,cooled to 0° C. and quenched by slow addition of 10 ml of saturatedNaHCO₃. The aqueous layer was extracted with CH₂Cl₂ (3×30 mL). Thecombined organic layers were washed with brine (2×30 mL) and dried overmagnesium sulfate and concentrated under vacuum. The residue waspurified by column chromatography (Silica gel, 20% EtOAc in hexanes) toprovide 630 mg of compound 6 (65%).

¹H NMR (300 MHz, CDCl₃) δ 0.87 (t, 3H, J=6.46 Hz), 1.26 (m, 16H), 1.58(p, 2H, J=6.79 Hz), 2.03 (s, 3H), 2.28 (b, 1H), 2.49-2.68 (m, 4H),2.82-2.92 (m, 2H), 4.20-4.34 (m, 4H), 6.69 (s, 1H), 6.89-7.05 (m, 3H);¹³C NMR δ 14.21, 14.25, 14.37, 22.92, 23.37, 25.48, 29.50, 29.63, 29.72,29.76, 30.39, 31.93, 32.13, 35.78, 40.33, 62.46, 62.79, 68.80, 126.04,128.81, 129.30, 132.85, 136.28, 140.66, 150.07, 167.63, 168.32, 169.42.

Example 6N-[2-Hydroxy-1-hydroxymethyl-1-(6-octyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-ethyl]-acetamide(7)

Lithium borohydride (2M solution in THF, 0.88 ml, 1.76 mmol) was addedto compound 6 (200 mg, 0.44 mmol) in 5 ml THF at 0° C. The reactionmixture was stirred at room temperature for 48 hours and diluted with 40ml ethyl acetate. The solution was washed with brine (2×20 mL) and driedover magnesium sulfate and concentrated under vacuum. The residue waspurified by column chromatography (Silica gel, 4% MeOH in CH₂Cl₂) toprovide 55 of mg compound 7 (33%).

¹H NMR (300 MHz, CDCl₃) δ 0.88 (t, 3H, J=6.56 Hz), 1.29 (m, 10H), 1.57(p, 2H, J=6.25 Hz), 1.94-1.98 (m, 2H), 2.05 (s, 3H), 2.33 (m, 1H), 2.51(t, 2H, J=7.32), 2.60-2.85 (m, 4H), 3.69 (d, 2H, J=11.61), 3.89 (dd, 2H,J=11.61 Hz, 7.25 Hz), 6.22 (s, 1H), 6.88-6.99 (m, 3H); ¹³C NMR δ 14.38,22.92, 24.20, 24.35, 29.52, 29.66, 29.73, 29.95, 30.32, 31.94, 32.14,35.78, 38.26, 63.55, 64.34, 64.46, 126.18, 128.85, 129.30, 133.06,136.22, 140.75, 172.40.

Example 72-Amino-2-(6-octyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-propane-1,3-diol(VPC104061)

A solution of compound 7 (53 mg, 0.14 mmol) and LiOH.H₂O (45 mg, 1.1mmol) in MeOH (3 ml), THF (1.5 ml) and water (3 ml) was stirred at 50°C. for 5 hours and diluted with ethyl acetate (20 ml). The solution waswashed with brine (2×10 mL) and dried over magnesium sulfate andconcentrated under vacuum. The residue was purified by columnchromatography (Silica gel, 50% MeOH in CH₂Cl₂) to provide 35 mg ofcompound VPC104061 (75%).

¹H NMR (300 MHz, CDCl₃) δ 0.88 (t, 3H, J=6.17 Hz), 1.29 (m, 10H), 1.56(p, 2H, J=6.17 Hz), 1.82-1.98 (m, 2H), 2.51 (t, 2H, J=6.95), 2.58-2.88(m, 5H), 3.19 (b, 4H), 3.61 (d, 2H, J=10.98), 3.73 (d, 2H, J=10.61 Hz),6.87-6.98 (m, 3H); ¹³C NMR δ 14.37, 22.93, 24.02, 29.32, 29.53, 29.70,29.75, 29.85, 30.26, 31.94, 32.14, 35.08, 39.58, 57.74, 66.13, 66.19,126.09, 128.81, 129.39, 133.28, 136.26, 140.64. MS (ESI) m/z 334.1[M+H]⁺.

Example 8 Phosphoric acidmono-[2-amino-2-(6-octyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-3-phosphonooxypropyl]ester(VPC104081)

Phosphorus pentoxide (2.0 g, 14 mmol) in phosphoric acid (85% in water,2 ml, 29 mmol) was added to VPC104061 (25 mg, 0.07 mmol). The mixturewas stirred at 100° C. for 2 hours and cooled to 0° C. The product wasprecipitated by adding water (14 mg, 37%). MS (ESI) m/z 494.4 [M+H]⁺.

Example 9 Amino-(6-octyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-acetic acid(8)

Compound 6 (300 mg, 0.65 mmol) was added to 12 M HCl (10 ml). Themixture was heated to reflux and MeOH (5 ml) was added until the mixturebecame homogenous. Reflux was continued for 2 hours until the startingmaterial was consumed as determined by thin layer chromatography (TLC).The reaction mixture was concentrated under reduced pressure andco-evaporated with MeOH and diethyl ether multiple times. The desiredcompound 8 was recrystallized from diethyl ether and hexanes to providea light brown solid and used directly for the next reaction.

Example 102-Amino-2-(6-octyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-ethanol(VPC104059)

The amino acid 8 prepared in Example 9 was added to a refluxing solutionof lithium aluminum hydride (62 mg, 1.63 mmol) in THF (10 ml). Thereaction mixture was heated at reflux for 12 hours, subsequently cooledto 0° C. and 10 M NaOH was added and stirred for 20 minutes. Ethylacetate (20 ml) was added and the mixture was filtered through Celiteand magnesium sulfate. The filtrate was concentrated under vacuum andpurified by column chromatography (Silica gel, 50% MeOH in CH₂Cl₂) toprovide 41 mg of the product, VPC104059 (21%, two steps).

¹H NMR (300 MHz, CDCl₃) δ 0.88 (t, 3H, J=6.39 Hz), 1.28 (m, 10H), 1.55(p, 2H, J=7.16 Hz), 1.67-2.11 (m, 3H), 2.48 (t, 2H, J=7.69), 2.56-2.83(m, 5H), 3.19 (b, 4H), 3.47-3.75 (m, 2H), 6.82-6.96 (m, 3H); ¹³C NMR δ13.34, 22.58, 26.45, 29.14, 29.21, 29.29, 29.47, 31.70 31.90, 32.26,35.40, 47.56, 125.64, 128.36, 128.86, 128.93, 133.19, 139.93, MS (ESI)m/z 303.9 [M+H]⁺.

Example 11 Phosphoric acidmono-[2-amino-2-(6-octyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-ethyl]ester(VPC104127)

Phosphorus pentoxide (1.5 g, 10.5 mmol) in phosphoric acid (85% inwater, 1.5 ml, 22 mmol) was added to VPC104059 (25 mg, 0.08 mmol). Themixture was stirred at 100° C. for 2 h and cooled to 0° C. The productwas precipitated by adding water (10 mg, 50%). MS (ESI) m/z 384.2[M+H]⁺.

Example 12 Synthesis of Structure (X)

The synthesis of an ether containing compound having formula IX isillustrated in Scheme 2 (FIG. 3). Keto-alcohol 1A, is converted to theketo-ether 1B, using standard reagents and techniques. The keto-ether ishalogenated to provide halo-ether 1C in a manner similar to Example 3.The halo-ether is alkylated to provide diester-ether 1D, in a mannersimilar to the procedure described in Example 4. The diester isconverted to ether-triol 1E, using standard reducing agents known in theart. The triol is converted to a diol and deprotected using standardmethods known in the art, to provide compound IX.

Example 132-Acetylamino-2-(6-octyl-1-hydroxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-malonicacid diethyl ester (A)

To a solution of sodium borohydride (75 mg, 2.00 mmol) in 5 ml ofethanol at room temperature is added2-acetylamino-2-(6-octyl-1-oxo-1,2,3,4-tetrahydro-naphthalen-2-yl)-malonicacid diethyl ester (compound 5) (1.00 g, 2.1 mmol) in 5 ml of ethanol.The reaction mixture is stirred at room temperature under argon for anadditional hour, quenched by the addition of water (20 mL) and methylenechloride (20 mL). The organic layer is removed and the aqueous layer isextracted with methylene chloride (2×20 mL). The combined organic layerswere washed with brine (2×20 mL) and dried over magnesium sulfate andconcentrated under vacuum. The residue is purified by columnchromatography (silica gel, 20% ethyl acetate in hexanes) to provide 755mg of compound A (75%).

Example 14 2-Acetylamino-2-(6-octyl-3,4-dihydro-naphthalen-2-yl)-malonicacid diethyl ester (B)

2-Acetylamino-2-(6-octyl-1-hydroxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-malonicacid diethyl ester (compound A, 755 mg, 1.58 mmol)) is dissolved inacetic anhydride (5 mL) followed at 0° C. by a catalytic amount offerric chloride (66 mg, 0.4 mmol). The reaction is stirred at 0° C. foran additional 2 hours, and 20 mL of diethyl ether is added. The reactionis carefully poured into 50 mL of ice cold water and the organic layerquickly separated. The organic layer is re-extracted with methylenechloride (2×20 mL) and the combined organic layers were washed once withbrine (20 mL) and dried over magnesium sulfate. The organic layer isconcentrated under vacuum and the residue is purified by columnchromatography (silica gel, 10% ethyl acetate in hexanes) to provide 458mg of compound B (60%). The complete synthesis is illustrated in FIG. 4.

Example 14 Sphingosine Kinase Assay

Recombinant sphingosine kinase type 2 (SPHK2) is prepared by forcing theexpression of the mouse or human recombinant enzyme by transfecting therelevant plasmid DNA into HEK293T cells. After about 60 hours, cells areharvested, broken and the non-microsomal (e.g., soluble) fraction isretained. The broken cell supernatant fluid containing the recombinantenzyme is mixed with test compounds (FTY720, AA151, VIII and XVIII)(5-50 micromolar) and γ-32P-ATP and incubated for 0.5-2.0 hours at 37°C. The lipids in the reaction mixture are extracted into an organicsolvent and displayed by normal phase thin layer chromatography. Theradio-labeled bands are detected by autoradiography, scraped from theplate and quantified by scintillation counting. In the histogram shown,(FIGS. 5A, 5B and 6) sphingosine was present at 15 μM, and the testcompounds were at a concentration of 50 μM, incubation time was 20minutes.

Example 15 GTPγS-35 Binding Assay

This assay illustrates agonist activation of G protein coupled receptors(GPCRs) in isolation. The assay forces expression concomitantly of arecombinant GPCR (e.g., the S1P1-5 receptor) and each of the threesubunits (typically, α-2, β-1, or γ-2) of a heterotrimeric G protein ina HEK293T cell by transfecting the cell with four plasmid DNAs encodingthe respective proteins. About 60 hours after transfection the cells areharvested, opened, and the nucleus discarded. The crude microsome isprepared from the remainder. Agonist (e.g., S1P) stimulation of thereceptor-G protein complex on the microsomes results in the exchange ofGTP for GDP on the α-subunit in a dose-dependent manner. The GTP-boundα-subunit is detected using a GTP analog (GTPγS-35), which is aradionuclide (sulfur-35) labeled phosphothionate that is not hydrolyzedto GDP. The microsomes with the adherent G proteins are collected byfiltration and the bound GTPγS-35 quantified in a liquid scintillationcounter. The assay yields relative potency (EC₅₀ values) and maximumeffect (efficacy, E_(max)). Antagonist activity is detected as rightwardshifts in the agonist dose-response curve in the presence of a fixedamount of antagonist. If the antagonist behaves competitively, theaffinity of the receptor/antagonist pair (K_(i)) can be determined.

The phosphorylated forms of compound VIII (a mixture of mono, XXVI, anddiphosphorylated, XXVII, isomers) and compound XXVIII are low potency,partial agonists at the S1P₃ receptor (See FIG. 8.). Compounds XXVI,XXVII and XXVIII are more potent at S1P₁ and less potent at S1P₃,relative to S1P. (See FIG. 7.) The assay was performed as described inDavis, M. D., J. J. Clemens, T. L. Macdonald and K. R. Lynch (2005) “S1PAnalogs as Receptor Antagonists” Journal of Biological Chemistry, vol.280, pp. 9833-9841.

Example 16 Lymphopenia Assay

Compounds (e.g., primary alcohols such as compound VIII) are dissolvedin 2% hydroxypropyl beta-cyclodextrin and introduced into groups of miceby oral gavage at doses from 0.01, 1.0 and 10 mg/kg body weight. After24 hours and 48 hours, the mice are lightly anesthetized and ca. 0.1 mlof blood is drawn from the orbital sinus. The number of lymphocytes (inthousands per microliter of blood; normal is 4-11) is determined using aHemavet blood analyzer. In the histogram showing compound VIII, the 100%value for the vehicle treated mouse was 7.5 at 24 hours; at 96 hours itwas 5. There were three mice/group, the strain was mixed sv129×C57BL/6.Active compounds (e.g., compound VIII and compound XXVIII) are dissolvedin acidified DMSO at 20 mM, and diluted 1:20 into 2% hydroxypropylbeta-cyclodextrin in water with mixing. This solution is introduced intomice by intraperitoneal (i.p.) injection at doses of 0.01, 1.0 and 10mg/kg body weight.

In FIG. 9 the results of an assay using compound VIII and compoundXXVIII administered via oral gavage are graphically illustrated. Thetotal lymphocyte counts (k/μl) 24 hours after an IV dose of compoundVIII is reported.

Compounds were dissolved in 2% hydroxypropyl β-cyclodextrin andadministered to sets of three, 12 week old C57B1/6 female mice by oralgavage (PO). At 18 hours post dosing, blood was drawn from the orbitalsinus and lymphocyte numbers were determined with a Hemavet bloodanalyzer. Doses were: compound XXIX (1 mg/kg), compound VIII (10 mg/kg),and FTY720 (0.3 mg/kg). Compound XXIX, 3,5-diphenyl-1,2,4-oxadiazole, isa S1P1 receptor agonist described by Li et al. (Journal MedicinalChemistry, vol. 48, p. 6169 (2005)) was used as a comparison. Theresults are illustrated in FIG. 10.

Example 17 Heart Rate Assay

Mice were dosed with compound VIII, FTY720 (intravenous, 3 mg/kg) orvehicle (2% hydroxypropyl beta-cyclodextrin) and heart rate measured at1 hour post dosing. Heart rate was captured in unrestrained, consciousanimals using the ECGenie™ system. The results are illustrated in FIG.11.

The invention should not be construed to be limited solely to the assaysand methods described herein, but should be construed to include othermethods and assays as well. Other methods which were used but notdescribed herein are well known and within the competence of one ofordinary skill in the art of chemistry, biochemistry, molecular biology,and clinical medicine. One of ordinary skill in the art will know thatother assays and methods are available to perform the proceduresdescribed herein.

The abbreviations used herein have their conventional meaning within theclinical, chemical, and biological arts. In the case of anyinconsistencies, the present disclosure, including any definitionstherein will prevail.

The disclosures of each and every patent, patent application, andpublication cited herein are expressly incorporated herein by referencein their entirety into this disclosure. Illustrative embodiments of thisdisclosure are discussed and reference has been made to possiblevariations within the scope of this disclosure. These and othervariations and modifications in the disclosure will be apparent to thoseskilled in the art without departing from the scope of the disclosure,and it should be understood that this disclosure and the claims shownbelow are not limited to the illustrative embodiments set forth herein.

1. A compound of formula I

wherein X¹, Y¹ and Z¹ are independently O, CR^(a), CR^(a)R^(b), N,NR^(c), or S; R¹ is hydrogen, halo (C₁-C₁₀)alkyl, (C₁-C₁₀)haloalkyl, or(C₁-C₁₀)alkoxy; R² is hydrogen, halo, (C₁-C₂₀)alkyl, (C₁-C₂₀)alkoxy;(C₂-C₂₆)alkoxyalkyl; (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl,(C₃-C₁₂)cycloalkyl, (C₆-C₁₀)aryl, (C₇-C₃₀)arylalkyl,(C₂-C₁₀)heterocyclic, and (C₅-C₁₀)heteroaryl; or R² can be a grouphaving formula II, III, IV, V, or VI;

where R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹²R¹³, and R¹⁴ are independently O, S, C,CR¹⁵CR¹⁶R¹⁷,C═O, N or NR¹⁸ R¹⁵, R¹⁶ and R¹⁷ are independently hydrogen,halo, (C₁-C₁₀)alkyl, (C₁-C₁₀)alkyl substituted with halo, hydroxy,(C₁-C₁₀)alkoxy, or cyano; and where R¹⁸ can be hydrogen or(C₁-C₁₀)alkyl; where at least one of R¹⁰, R¹¹R¹², R¹³, or R¹⁴ is aheteroatom; where Z² is (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, substitutedalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, aryl, alkyl substituted aryl,arylalkyl, or aryl substituted arylalkyl; wherein the alkyl groups of Z²are optionally substituted with 1, 2, 3, or 4 substituent groups wherethe substituent groups independently are halo, (C₁-C₁₀)alkoxy or cyano;

indicates one or more optional double bonds; wherein Y² is O, C═O, orCH₂; W¹ is a bond or —CH₂—CH₂—CH₂—; W² is a bond or —CH₂— and m is 1, 2,or 3, or (C═O)(CH₂)₁₋₅ and m is 1; and n is 0, 1, 2, or 3; each

represents an optional double bond; R³ is hydrogen, (C₁-C₁₀)alkyl, or(C₁-C₁₀)alkoxy; R⁴ is hydroxyl (—OH), phosphate (—OPO₃H₂), phosphonate(—CH₂PO₃H₂), or alpha-substituted phosphonate; R^(a), R^(b), and R^(c)are independently hydrogen, or (C₁-C₁₀)alkyl; wherein the alkyl groupsof R¹ are optionally substituted with 1, 2, 3, or 4 substituent groupswhere the substituent groups independently are halo, (C₁-C₁₀)alkoxy orcyano; wherein any of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heterocyclic, or heteroaryl groups of R² are optionally substituted with1, 2, 3, or 4 substituent groups where the substituent groupsindependently are oxo (═O), imino (═NR^(d)), (C₁-C₁₀)alkyl,(C₁-C₁₀)alkoxy, or C₆-aryl; or wherein one or more of the carbon atomsin the R² alkyl groups can be independently replaced with non-peroxideoxygen, sulfur or NR^(c); the alkyl groups of R³ are optionallysubstituted with 1, or 2 hydroxy groups; and R^(d) is hydrogen, or(C₁-C₁₀)alkyl; or a pharmaceutically acceptable salt or ester thereof.2. The compound of claim 1, wherein R¹ is hydrogen, fluorine, chlorine,bromine, trifluoromethyl, methoxy, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,(C₁-C₆)alkyl substituted with, alkoxy or cyano, alkyl-substituted aryl,aryl-substituted alkyl, or aryl-substituted arylalkyl.
 3. The compoundof claim 2, wherein R¹ is hydrogen, trifluoromethyl, or —CH₂CF₃.
 4. Thecompound of claim 2, wherein R¹ is benzyl, phenylethyl, or methylbenzyl.
 5. The compound of claim 1, wherein R² comprises—CH₂—CH₂—O—CH₂—CH₂—O—.
 6. The compound of claim 1, wherein R² is


7. The compound of claim 6, wherein R² is:

where Y³ is (CH₃)₃C—, CH₃CH₂(CH₃)₂C—, CH₃CH₂CH₂—, CH₃(CH₂)₂CH₂—,CH₃(CH₂)₄—CH₂—, (CH₃)₂CHCH₂—, (CH₃)₃CCH₂—, CH₃CH₂O—, (CH₃)₂CHO—, orCF₃CH₂CH₂— or a group having the formula:


8. The compound of claim 7, wherein R² is:


9. The compound of claim 8, wherein R² is:


10. The compound of claim 6, wherein R² is:


11. The compound of claim 10, wherein R² is


12. The compound of claim 1, wherein R² has formula IV


13. The compound of claim 12, wherein R² is


14. The compound of claim 1, wherein R² is (C₁-C₁₀)alkyl,(C₂-C₁₀)alkenyl and (C₂-C₁₄)alkynyl, (C₁-C₁₀)alkoxy or(C₂-C₁₆)alkoxyalkyl.
 15. The compound of claim 14, wherein R² is(C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy or (C₂-C₁₂)alkoxyalkyl.
 16. The compoundof claim 15, wherein R² is methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, octyl, trifluoromethyl, trifluoroethyl, trifluoromethoxy,trifluoroethoxy, methoxy, ethoxy, propoxy, butoxy, pentoxy, heptoxy, oroctoxy.
 17. The compound of claim 1, wherein each of X¹, Y¹ and Z¹ isCH₂.
 18. The compound of claim 1, wherein R³ is hydrogen, methyl,hydroxymethyl, ethyl, hydroxyethyl, propyl, or isopropyl.
 19. Thecompound of claim 18, wherein R³ is hydrogen, methyl, hydroxymethyl,ethyl, or hydroxyethyl.
 20. The compound of claim 1, having the formula


21. The compound of claim 20, having the formula:


22. A method for prevention or treatment of a pathological condition orsymptom in a mammal, wherein the activity of sphingosine 1-phosphatereceptors is implicated and agonism of such activity is desired,comprising administering to said mammal an effective amount of acompound of claim
 1. 23. The method of claim 21, wherein thepathological condition is an autoimmune disease.
 24. The method of claim22, wherein the autoimmune disease is uveitis, type I diabetes,rheumatoid arthritis, inflammatory bowel diseases, or multiplesclerosis.
 25. The method of claim 24, wherein the autoimmune disease ismultiple sclerosis.
 26. The method of claim 22, wherein the preventionor treatment of a pathological condition is altering lymphocytetrafficking.
 27. The method of claim 26, wherein altering lymphocytetrafficking provides prolonged allograft survival.
 28. The method ofclaim 27, wherein the allograft is for transplantation.
 29. A method forprevention or treatment of a pathological condition or symptom in amammal, wherein the activity S1P lyase implicated and inhibition of theS1P lyase is desired, comprising administering to said mammal aneffective amount of a compound of claim 1.